This invention relates generally to synthetic oligonucleotide compounds. More specifically, this invention relates to cleavage of oligonucleotides from solid supports and deprotection of oligonucleotides.
Oligonucleotides are essential reagents in many important molecular biology experiments, assays and information gathering operations, such as the polymerase chain reaction (PCR), diagnostic probes, single nucleotide polymorphism (SNP) detection, and genomic sequencing. The benefits of conducting the synthesis of oligonucleotides by the sequential addition and covalent attachment of monomeric units onto a solid support is well appreciated. In particular, the method of Caruthers is highly optimized and almost universally adopted (U.S. Pat. Nos. 4,458,066 and 4,973,679). The vast majority of the millions of oligonucleotides consumed each year are prepared by automated synthesis with phosphoramidite nucleoside monomers (Beaucage (1992) Tetrahedron Lett. 22:1859-62; U.S. Pat. No. 4,415,732).
Conducting chemical reactions on solid supports has several practical advantages: (i) excess reagents and soluble by-products can be easily removed and separated by simple washing and filtration steps, (ii) dispensing, manipulating, organizing the parallel production of many oligonucleotides is facilitated, and (iii) reactions can be scaled up or down for economy and ease of handling.
Many applications utilize oligonucleotides with a covalently attached label. Labels may impart some function, e.g. affinity, detection, or other physical property. Oligonucleotide labels often have reactive functionality, which may preferably be protected to minimize side reactions and modifications.
Upon completion of synthesis, the solid support-bound oligonucleotide is removed from the support by chemical cleavage of the covalent linkage between the oligonucleotide and the solid support, and deprotected to remove all remaining protecting groups from the oligonucleotide. The steps of cleavage and deprotection may be concurrent and conducted with the same reagent. Alternatively, cleavage and deprotection may be conducted at different temperatures and with different reagents.
Typically, cleavage of the oligonucleotide (20 nmole to 1 xcexcmole) from the solid support is performed in the synthesis column at room temperature using about 1 to 3 ml concentrated ammonium hydroxide NH4OH (about 28-30% NH3 in water). Cleavage of the typical ester linkage at the 3xe2x80x2 terminus of the oligonucleotide is complete in about one hour under these conditions. While the linkage between the oligonucleotide and the solid support is cleaving, ammonium hydroxide is also removing the 2-cyanoethyl groups from the intemucleotide phosphates and the nucleobase protecting groups. Depending on the nucleobase and the type of protecting groups, deprotection (removal of protecting groups) of the oligonucleotide requires approximately 1 to 8 hours at 55xc2x0 C. treatment with concentrated ammonium hydroxide.
Alternatively, cleavage and deprotection may be conducted with anhydrous amines (U.S. Pat. No. 5,750,672), methylamine (U.S. Pat. Nos. 5,348,868 and 5,518,651), hydrazine and ethanolamine (Polushin (1991) Nucleic Acids Res. Symposium Series No. 24, p. 49-50; Polushin (1994) Nucleic Acids Res. 22:639-45)
A typical post-synthesis, cleavage/deprotection routine on automated DNA synthesizers (e.g. Models 392, 394, 3948, Applied Biosystems, Foster City, Calif.) delivers concentrated ammonium hydroxide through the synthesis column after completion of oligonucleotide synthesis and allows it to stand in the column for about one hour, with periodic deliveries of more ammonium hydroxide and collection of the eluant in a vessel. The vessel containing the cleaved and partially deprotected oligo nucleotide can then be transferred to a heating device to complete deprotection. Alternatively, the nucleobase protecting groups may be sufficiently labile to not require further heating to yield a fully deprotected oligonucleotide. The ammonium hydroxide is removed under vacuum or in a stream of air or inert gas. The crude oligonucleotide may be purified by various methods, including hydrophobic cartridge purification, reverse-phase HPLC, polyacrylamide gel electrophoresis, and precipitation. For some applications, the crude oligonucleotide may be pure enough to perform adequately.
After completion of cleavage of the oligonucleotides from the support, the remaining protecting groups are removed by incubation in the ammonium hydroxide solution at either room temperature or with heating, e.g. 55xc2x0 C. for 6-24 hours. Alternatively, oligonucleotides can be cleaved and/or deprotected with ammonia, or other amines, in the gas phase whereby the reagent gas comes into contact with the oligonucleotide while attached to, or in proximity to, the solid support (U.S. Pat. Nos. 5,514,789; 5,738,829).
The particular cleavage and deprotection protocol used in any situation is largely determined by protecting groups employed on the nucleobases, the intemucleotide phosphorus, the sugars, 3xe2x80x2 or 5xe2x80x2 terminus, and any covalently attached label. The first generation set of nucleobase protecting groups utilized in the phosphodiester method of synthesis includes benzoyl (bz) and isobutyryl (ibu) protecting groups, utilized as adenosine Abz, cytosine Cbz and guanosine Gibu (Schaller (1963) J. Amer. Chem. Soc. 85, 3821-3827 and Buchi (1972) J. Mol. Biol. 72:251). Generally, thymidine T is not protected.
It is known that certain side-reactions occur during the cleavage and deprotection reactions. Modifications of the nucleobases, internucleotide phosphate groups, and pendant amino groups have been characterized (Chang (1999) Nucleosides and Nucleotides 18:1205-1206; Manoharan (1999) Nucleosides and Nucleotides 18:1199-1201). Acrylonitrile, released from deprotection of the internucleotide phosphate groups, may form adducts on the nucleobases, labels, or other sites (EP 1028124; WO 0046231; Eritja (1992) Tetrahedron 48:4171-82; Wilk (1999) J. Org. Chem. 64:7515-22). Other impurities are uncharacterized, but known to detract from the purity of oligonucleotides and cause loss of performance. Where deprotection of protecting groups is incomplete, oligonucleotides may hybridize with lower specificity or affinity, leading to mispriming or mutagenicity.
New reagents and methods for cleavage and deprotection of oligonucleotides are desirable. Certain protecting groups may not be compatible with deprotection reagents or automated synthesizers and protocols, leading to modifications. Certain labels, e.g. those with extended conjugation or reactive functionality, may lead to modifications of the labels or the oligonucleotide during the cleavage and deprotection steps. Reagents and methods which minimize or eliminate side reactions and modifications are desirable.
The present invention provides a process for the removal of protecting groups, i.e. deprotection, from chemically synthesized oligonucleotides. In one embodiment, the invention provides reagents suitable for use in such a process, and kits incorporating such reagents in a convenient, ready-to-use format. By use of the process and reagents of the invention, side-reactions leading to certain impurities that contaminate the synthesized oligonucleotides can be minimized.
In a first aspect, the invention provides a method for deprotection of an oligonucleotide by reacting a protected oligonucleotide with a deprotection reagent wherein the deprotection reagent comprises an active methylene compound and an amine reagent. The active methylene compound has the structure: 
The substituent EWG is an electron-withdrawing group selected from nitro, ketone, ester, carboxylic acid, nitrile, sulfone, sulfonate, sulfoxide, phosphate, phosphonate, nitroxide, nitroso, trifluoromethyl and aryl groups substituted with one or more nitro, ketone, ester, carboxylic acid, nitrile, sulfone, sulfonate, sulfoxide, phosphate, phosphonate, nitroxide, nitroso, and trifluoromethyl. The substituent R is selected from hydrogen, C1-C12 alkyl, C6-C20 aryl, heterocycle and electron-withdrawing group. The amine reagent may be aqueous ammonium hydroxide, aqueous methylamine, or anhydrous C1-C6 alkylamine. In addition to an active methylene compound and an amine reagent, the deprotection reagent of the invention may include water or an alcohol solvent. Protecting groups are removed from the oligonucleotide by treatment with the deprotection reagent.
The oligonucleotide may be covalently attached to a solid support through a linkage. The oligonucleotide may be cleaved from the solid support either before, during, or after the protecting groups are removed. The solid support may be an organic polymer or inorganic. The solid support may be a membrane or frit which allows the deprotection reagent to pass through.
The solid support may be confined in a column or other enclosure which has inlet and outlet openings for the deprotection reagents to pass or flow through. The columns may be configured in a variety of formats, including holders of many columns, e.g. 96- or 384-well microtitre plate formats. A plurality of oligonucleotides in a holder may be deprotected concurrently or separately through discriminate or indiscriminate delivery or exposure to the deprotection reagents.
Oligonucleotides which may be deprotected by the deprotection reagents of the invention include nucleic acid analogs. Oligonucleotides may bear one or more covalently attached labels such as a fluorescent dye, a quencher, biotin, a mobility-modifier, and a minor groove binder.
In a second aspect, the invention provides a method for deprotection of an oligonucleotide by first wetting the protected oligonucleotide covalently attached to the solid support with an active methylene compound and a solvent, and then reacting the protected oligonucleotide with an amine reagent. The amine reagent may be in liquid or gas phase; aqueous or anhydrous, e.g. aqueous ammonium hydroxide, ammonia gas or a C1-C6 alkylamine.
In a third aspect, the invention includes an oligonucleotide deprotection reagent wherein the deprotection reagent comprises an active methylene compound and an amine reagent. The active methylene compound has the structure: 
The substituent EWG is an electron-withdrawing group selected from nitro, ketone, ester, carboxylic acid, nitrile, sulfone, sulfonate, sulfoxide, phosphate, phosphonate, nitroxide, nitroso, trifluoromethyl and aryl groups substituted with one or more nitro, ketone, ester, carboxylic acid, nitrile, sulfone, sulfonate, sulfoxide, phosphate, phosphonate, nitroxide, nitroso, and trifluoromethyl. The substituent R is selected from hydrogen, C1-C12 alkyl, C6-C20 aryl, heterocycle and electron-withdrawing group. The active methylene compound may be 1 to 10% by volume of the deprotection reagent. The deprotection reagent may further include an alcohol solvent which is 1 to 30% by volume of the reagent.
In a fourth aspect, the invention includes deprotected oligonucleotides deprotected by the deprotectibn reagents of the invention.